Three flavonoid glycosides including quercetin-3-O-[2''-O-(6'''-O-E-sinapoyl)--D-glucopyranosyl]--D-glucopyra noside(I), quercetin-3-O-[2''-O-(6'''-O-E-feruloyl)--D-glucopyranosyl]--D-glucopyra noside(II) and quercetin-3-O-[2''-O-(6'''-O-E-feruloyl)--D-glucopyranosyl]--D-galactopy ranoside(III) were isolated and purified from Hedyotis diffusa Willd by high-speed counter-current chromatography (HSCCC). This run was carried out with a two-phase solvent system composed of n-hexane-ethyl acetate-n-butanol-methanol-1.0% acetic acid (1:1:3.5:1:4.5, v/v) by eluting the lower phase as the mobile phase with a flow-rate at 2.0ml/min. Consequently, 29.6mg of I, 35.1mg of II, 41.3mg of III with purities of over 95% were obtained from 200mg of the crude extracts in a single run in less than 130min. The structure of the isolated compounds was confirmed by MS, 1H NMR, and 13C NMR analysis.